Epstein-Barr Virus Associated Gastric Cancer: a Histopathologic Study with Comprehensive Molecular Profiling

Background: A subset of gastric cancers (GCs) is linked to Epstein-Barr virus (EBV) infection. This study aims to characterize the histopathological and molecular features of EBV-associated GCs (EBV-GCs), focusing on predictive biomarkers and genomic and transcriptomic analysis. Design: A total of 38 EBV-GCs were collected from five institutions. The presence of EBV was confirmed with in-situ hybridization. Immunohistochemistry for HER2, PD-L1, CLDN18 and MMR proteins were performed. Genomic and transcriptomic profile was assessed using AmoyDX Master panel that at the DNA level can identify SNV, InDels, and CNV on 571 hot genes, microsatellite status, TMB, and HRD, while on the RNA level, in addition to fusions on 45 hot genes, it also quantifies the expression of 2396 cancer-related transcripts. Results: Most EBV-GCs were diagnosed as pT3/pT4 (69%), were located in the corpus/fundus (50%) and were associated with intestinal metaplasia (63%). Six EBV-GCs (16%) were associated with dysplasia. The following histotypes were identified: carcinoma with lymphoid stroma (CLS; 63%), tubular high-grade (16%), mixed (13%) and tubular low-grade (8%). MMR deficiency and HER2 positivity were each observed in 5% of cases, while all tumors had a PD-L1 Combined Positive Score ≥ 10. CLDN18 moderate/ strong expression in ≥75% of cancer cells was detected in 67% of cases, though no clinicopathological or molecular associations were identified. The most frequently altered genes were PIK3CA (35%), AIRD1A (15%), APC (9%), CTNNB1(9%), KTM2C (9%) and KTM2D (9%). TMB-high status was found in 20.5% of cases. A preliminary transcriptomic analysis revealed a differential expression between CLS and the other histotypes. A total of 99 transcripts were found to be deregulated (Log2FC>1.5; Log2FC<- 1) among the two subgroups, including EBV-related transcripts, transcripts coding for oncoproteins, tumor suppressor proteins, cytokines and chemokines, transcripts coding for anti-cancer immune response regulators already druggable and novel regulators. Conclusions: EBV-GCs are a discrete pathologic and molecular entity. EBV-GCs are located mainly in the corpus/fundus, with common histotypes like CLS. Key molecular features include CLDN18 and PD-L1 positivity, frequent PIK3CA mutations, and TMB-high. Transcriptomic findings offer a foundation for further exploration into the molecular underpinnings of CLS and its distinctiveness from other histotypes, which may lead to improved therapeutic strategies.